Ulcerative proctitis, rectal prolapse, and intestinal pseudo-obstruction in transgenic mice overexpressing hepatocyte growth factor/scatter factor

Hepatocyte growth factor/scatter factor (HGF/SF) can stimulate growth of gastrointestinal epithelial cells in vitro; however, the physiological role of HGF/SF in the digestive tract is poorly understood. To elucidate this in vivo function, mice were analyzed in which an HGF/SF transgene was overexpressed throughout the digestive tract. Nearly a third of all HGF/SF transgenic mice in this study (28 of 87) died by 6 months of age as a result of sporadic intestinal obstruction of unknown etiology. Enteric ganglia were not overtly affected, indicating that the pathogenesis of this intestinal lesion was different from that operating in Hirschsprung's disease. Transgenic mice also exhibited a rectal inflammatory bowel disease (IBD) with a high incidence of anorectal prolapse. Expression of interleukin-2 was decreased in the transgenic colon, indicating that HGF/SF may influence regulation of the local intestinal immune system within the colon. These results suggest that HGF/SF plays an important role in the development of gastrointestinal paresis and chronic intestinal inflammation. HGF/SF transgenic mice may represent a useful model for the study of molecular mechanisms associated with a subset of IBD and intestinal pseudo-obstruction. Moreover, our data identify previously unappreciated side effects that may be encountered when using HGF/SF as a therapeutic agent.     

BAG1 over-expression in brain protects against stroke

The co-chaperone BAG1 binds and regulates 70 kDa heat shock proteins (Hsp70/Hsc70) and exhibits cytoprotective activity in cell culture models. Recently, we observed that BAG1 expression is induced during neuronal differentiation in the developing brain. However, the in vivo effects of BAG1 during development and after maturation of the central nervous system have never been examined. We generated transgenic mice over-expressing BAG1 in neurons. While brain development was essentially normal, cultured cortical neurons from transgenic animals exhibited resistance to glutamate-induced, apoptotic neuronal death. Moreover, in an in vivo stroke model involving transient middle cerebral artery occlusion, BAG1 transgenic mice demonstrated decreased mortality and substantially reduced infarct volumes compared to wild-type littermates. Interestingly, brain tissue from BAG1 transgenic mice contained higher levels of neuroprotective Hsp70/Hsc70 protein but not mRNA, suggesting a potential mechanism whereby BAG1 exerts its anti-apoptotic effects. In summary, BAG1 displays potent neuroprotective activity in vivo against stroke, and therefore represents an interesting target for developing new therapeutic strategies including gene therapy and small-molecule drugs for reducing brain injury during cerebral ischemia and neurodegenerative diseases.     

Genetic mapping of genes regulating the thymus size in back-cross rats between the laboratory BUF/Mna strain and the MITE strain derived from the wild rat, Rattus norvegicus

The thymoma prone BUF/Mna (B) rat is a useful model for Studying the genes responsible for thymus enlargement during the stage of young growth. Among the strains of rats, B rats have the largest thymuses at al stages of life. A locus, Ten-1 , which contributes to thymus enlargement in back-cross (BC) rats between the B and WKY/NCrj (W) strains, was mapped on chromosome 1. To determine the precise location of the bus, (B×(B×MITE)F1) BC rats were generated by crossing the B strain with the Inbred MITE (M) strain, which was established from captured, Japanese wild rats, and were examined by linkage study using polymerase chain reaction with 67 microsatellite markers. Linkages with thymus enlargements were found In genotypes of seven markers, BSIS, LSN, MYL2, IGF2, PBPC2, D1Mgh11 , and D1Mit6 , by X^2-test and Student's t -test, which confirmed the presence of the genetic locus associated with thymus enlargement, Ten-1 , in this region. Paradoxically, a suppressive locus, Tsu-1 , to thymus enlargement was also found on chromosome 3, showing linkages of phenotype of the small thymus with genotypes of SCN2A, CAT D3Mit16 , and D3Mit13 . By analyses of mapmaker/exp and mapmaker/qtl, Ten-1 was mapped at 4.6 cM proximal from IGF2 locus on chromosome 1 and Tsu-1 at 4.0 cM proximal from CAT locus on chromosome 3, respectively.     

Calcineurin-mediated BAD Ser155 dephosphorylation in ammonia-induced apoptosis of cultured rat hippocampal neurons

We previously reported that ammonia induced apoptosis in cultured rat hippocampal neurons with moderate increases in the intracellular calcium concentration and decreases in phospho-BAD levels. Since this suggested the involvement of calcineurin in the apoptosis, the effects of calcineurin inhibitors, 1 microM cyclosporin A and 1 microM FK506, on the ammonia-induced neuronal apoptosis were tested. Both of the inhibitors abolished the neuronal apoptosis assessed by double staining with Hoechst 33258 and anti-neurofilament antibody, and the ammonia-induced decrease in phospho-BAD Ser(155) level. Thus, calcineurin appeared to be involved in the dephosphorylation of BAD at the sites including Ser(155) in ammonia-induced apoptosis.     

Expression of EGF, EGF-receptor, p53, v-erb B and ras p21 in colorectal neoplasms by immunostaining paraffin-embedded tissues

Immunohistochemical studies were performed to clarify the significance of the expression or overexpression of epidermal growth factor (EGF), EGF-receptor (EGFR), p53, v- erb B, ras p21 in 23 cases each of tubular adenoma and adenocarcinoma. The expression of EGF, EGFR, p53, v- erb B, and ras p21 in paraffin-embedded tissues, from 46 patients with colorectal tumors (adenoma: 23 cases; 14 mild dysplasia, six moderate dysplasia, three severe dysplasia, adenocarcinoma: 23 cases; 17 well differentiated, two moderately differentiated, three poorly differentiated, one mucinous carcinoma was analyzed immunohistochemically using anti-EGF, EGFR, p53, v- erb B and ras p21 antibodies. The EGF and ras p21 tended to express more strongly in carcinoma cases than in the adenoma cases, and in severe and moderate dysplasia than in mild dysplasia (EGF: stained positive in five adenomas [21.74%] and 17 adenocarcinomas [73.91%]; ras p21: stained positive in six adenomas [26.09%] and 14 adenocarcinomas [60.87%]. The EGFR stained positive in two adenomas (8.70%) and two adenocarcinomas (8.70%). The p53 and v- erb B showed positive staining only in the carcinoma cases (p53: stained positive in four cases [17.39%]; v- erb B: stained positive in eight cases [34.78%]). This study suggests that these factors seem to have some role in the progression of colon neoplasms. It suggests that genetic alteration is not always equal to the overexpression of protein products, but that it reflects them well, and that the staining makes some contribution to differential diagnosis in colorectal neoplasms.     

A second group of hepatitis C viruses

cDNA clone 11–7 was isolated by immunoscreening a cDNA library that was prepared from a pooled plasma of non-A non-B hepatitis (NANBH) patients using expression vector gt 11. This cDNA corresponds to known nucleotide positions 3983–4745 of the genome of hepatitis C virus (HCV). This clone was used as a probe for screening the HCV-related cDNAs in a cDNA library similarly prepared by using gt 10. As a result, six more cDNA clones were isolated and analyzed for their nucleotide sequences. The results strongly suggested that there are at least two groups of HCV, group I and group II. According to our classification, the prototype HCV and clone 11–7 belong to group I HCV, and their nucleotide and deduced amino acid sequences were diverged from those of group II HCV. Genetic variation observed in the nucleotide and the amino acid sequences between the two groups resembles that in the NS3 region of the genome between Japanese encephalitis virus and West Nile fever virus. Polypeptides produced inEscherichia coli carrying a clone 11–7 or a group II cDNA clone E reacted with antibodies in the blood of 12 or 4 out of 14 individual chronic NANBH patients, respectively. Our data clearly indicate the existence of a second group of HCV.     

Relationship between neurovirulence and temperature sensitivity of an attenuated western equine encephalitis virus

Summary An attenuated strain of western equine encephalitis virus which arose spontaneously during a persistent infection was compared with the virulent parent strain for its growth potential over the temperature range 32° to 42° C. At 42° C, the attenuated strain proved unable to grow in a one-step growth experiment and failed to produce plaques. The temperature sensitivity was found to be due not to inactivation at 42° C of the attenuated virus particles, but to some temperature sensitive step occurring late in the replicative cycle. When the attenuated strain was passaged at 41 °C and clones selected for growth at this temperature, the resulting population did not regain the neurovirulence of the wild-type virus. In contrast, when virulent-revertants were selected from the attenuated, temperature-sensitive virus, temperature sensitivity remained unchanged. These findings indicated a lack of covariation of the temperature sensitivity and neurovirulence of western equine encephalitis virus, and suggest that mutation to avirulence and temperature sensitivity probably occurred independently.     

Clinical usefulness of urinary diacetylpolyamines as novel tumor markers

N1,N12-Diacetylspermine(DiAcSpm) and N1,N8-diacetylspermidine(DiAcSpd) are excreted in the urine of healthy persons as minor components of urinary polyamine, with small individual variations in amount. They are promising tumor markers, since their excretion is frequently elevated in patients with various types of cancers. DiAcSpm is sensitive in cancer detection, while DiAcSpd is highly specific for cancer. Diacetylpolyamines were initially characterized and determined by HPLC fractionation, followed by enzymatic detection. More recently, antibodies highly specific for DiAcSpm and DiAcSpd were developed, and an ELISA system applicable to the determination of urinary DiAcSpm was established. Measurement of urinary DiAcSpm using this ELISA system revealed that DiAcSpm is a more sensitive tumor marker than CEA, CA19-9 and CA15-3 for colon and breast cancers. More importantly, DiAcSpm efficiently detects patients at early stages. On the other hand, CEA, CA19-9 and CA15-3 are quite insensitive for early stage cancers. The urinary DiAcSpm level tends to remain low even in tumor-bearing individuals when their cancerous lesions remain static, while it rises rapidly concomitant with recurrence. DiAcSpm may serve as a prognostic indicator and marker for recurrence of prostate and colon cancers. Diacetylpolyamines may turn out to be general tumor markers, since active proliferation of any cancer tissues would likely be accompanied by activation of polyamine metabolism.